Abstract：
To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene.
E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS).
Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline.
Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.
E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS).
Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline.
Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.
摘要：
目的：阐明由tet（A）变异基因介导的大肠埃希菌对替加环素的耐药机制。
方法：E.大肠杆菌573菌株携带质粒携带的tet（A）变异基因，暂时指定为TET(A)TIG，这导致替加环素敏感性降低（MIC0.5mg/L）。当暴露于浓度增加的替加环素（0.25-8mg/L）时，获得了以2、4和8mg/L生长的突变体并进行了测序。通过WGS和定量PCR(qPCR)测定突变体中质粒和tet(A)TIG相对于染色体DNA的拷贝。通过RT-qPCR评估突变体中tet(A)TIG的表达。通过S1-PFGE和Southern印迹杂交观察携带tet(A)TIG的质粒。PCR用于检测携带tet（A）TIG的非常规可环化结构（UCS）。
结果：在替加环素存在下选择的大肠杆菌突变体中观察到最大MIC为16mg/L的替加环素抗性。与亲本菌株相比，在2、4和8mg/L替加环素存在下，突变体中tet（A）TIG的相对拷贝数和转录水平显着增加，分别。随着替加环素选择压力的增加，突变体中携带tet（A）TIG的质粒大小增加，与带有tet（A）TIG的ΔTnAs1侧翼UCS的串联扩增数相关。这些串联扩增在不存在替加环素的情况下是不稳定的。
结论：替加环素抗性是由于大肠杆菌中ΔTnAs1侧翼的tet（A）TIG携带质粒片段的串联扩增。在存在/不存在替加环素的情况下串联扩增物的获得/损失代表了细菌在替加环素存在下存活的经济方式。
方法：E.大肠杆菌573菌株携带质粒携带的tet（A）变异基因，暂时指定为TET(A)TIG，这导致替加环素敏感性降低（MIC0.5mg/L）。当暴露于浓度增加的替加环素（0.25-8mg/L）时，获得了以2、4和8mg/L生长的突变体并进行了测序。通过WGS和定量PCR(qPCR)测定突变体中质粒和tet(A)TIG相对于染色体DNA的拷贝。通过RT-qPCR评估突变体中tet(A)TIG的表达。通过S1-PFGE和Southern印迹杂交观察携带tet(A)TIG的质粒。PCR用于检测携带tet（A）TIG的非常规可环化结构（UCS）。
结果：在替加环素存在下选择的大肠杆菌突变体中观察到最大MIC为16mg/L的替加环素抗性。与亲本菌株相比，在2、4和8mg/L替加环素存在下，突变体中tet（A）TIG的相对拷贝数和转录水平显着增加，分别。随着替加环素选择压力的增加，突变体中携带tet（A）TIG的质粒大小增加，与带有tet（A）TIG的ΔTnAs1侧翼UCS的串联扩增数相关。这些串联扩增在不存在替加环素的情况下是不稳定的。
结论：替加环素抗性是由于大肠杆菌中ΔTnAs1侧翼的tet（A）TIG携带质粒片段的串联扩增。在存在/不存在替加环素的情况下串联扩增物的获得/损失代表了细菌在替加环素存在下存活的经济方式。
